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Stronger cytotoxicity in CTLs with granzyme B and porforin was induced by Ganoderma lucidum polysaccharides acting on B16F10 cells
Biomedicine & Preventive Nutrition 2 (2012) 113–118
Available online at
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Original article
Stronger cytotoxicity in CTLs with granzyme B and porforin was induced by Ganoderma lucidum polysaccharides acting on B16F10 cells Li-Xin Sun a,b , Zhi-Bin Lin a , Xin-Suo Duan b , Jie Lu b , Zhi-Hua Ge b,∗ , You-Xin Song b , Xue-Jun Li a , Min Li a , En-Hong Xing b , Ning Yang b , Wei-Dong Li a,∗ a b
Department of Pharmacology, Peking University Health Science Centre, School of Basic Medical Sciences, Beijing 100191, China Affiliated Hospital of Chengde Medical College, Chengde 067000, Hebei Province, China
a r t i c l e
i n f o
Article history: Received 24 December 2011 Accepted 25 January 2012 Keywords: Ganoderma lucidum polysaccharides Tumor CTL Granzyme B Porforin Cytotoxicity Tumorigenesis
a b s t r a c t Cytotoxic T lymphocytes (CTLs) exert cytotoxicity against tumor cells with granzyme B and porforin (two important components for cytotoxicity). One main reason for the limitation of clinical success in tumor immunotherapy is tumor cell inefficacy in inducing sufficient immune responses, such as efficient CTLs, and subsequently sufficient granzyme B and porforin activity because of weak immunogenicity of the tumor cells. It is therefore important to boost tumor cells to induce efficient CTLs and sufficient granzyme B and porforin. We suggest that Ganoderma lucidum polysaccharides (Gl-PS), with multiple bioactivities have this potential. We have shown that after incubation with Gl-PS, B16F10 melanoma cells, which are deficient in antigen presentation, promoted cytotoxicity of CTLs against B16F10 cells, induced more granzyme B and porforin in CTLs, decreased the in vivo incidence of tumorigenesis 15 days after inoculation and prolonged the latency of tumorigenesis 21 days after inoculation, demonstrating that the effects of Gl-PS on B16F10 cells induced stronger immune responses against tumor cells. © 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction The immune system has the potential to recognize and attack tumor cells by various means involving different effector cells. Lymphocytes play an important role in tumor control and cytotoxic T lymphocytes (CTLs) are most of the effector cells. There are two pathways (cell death receptor-mediated pathway and granule exocytosis pathway), by what CTLs effect cytotoxicity against the target cells [1]. Of the two pathways, the granule exocytosis pathway is the principal pathway and involves granzyme B and porforin, which are two of the most important components exerting cytotoxicity against target cells [2]. However, despite the potential to eliminate tumor cells, T cells fail to do so because cancer cells have various strategies to avoid rejection by the host immune system [3], thereby inefficient induction of CTLs and insufficient production of granzyme B and porforin in lymphocytes occurs in cancer patients. Indeed, cancer immunotherapy would benefit by boosting cancer cells to induce efficient CTLs and sufficient granzyme B and porforin. Ganoderma lucidum polysaccharides (Gl-PS) may be one means by which to boost cancer cells.
∗ Corresponding authors. Tel.: +86 10 8280 2798; fax: +86 10 8280 1686. E-mail addresses: [email protected] (Z.-H. Ge), [email protected] (W.-D. Li). 2210-5239/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.bionut.2012.01.001
Gl-PS is an extract from G. lucidum, which has been widely used in China for centuries to promote longevity and improve vigour, without appreciable adverse effects [4,5]. Like some other macromolecules [6–12], Gl-PS has anti-tumor and immunomodulatory effects. Over the past two decades, numerous studies have demonstrated the multiple biological activities of Gl-PS, including immunomodulatory and anti-tumor activities [5,13,14]. The anti-tumor effect of Gl-PS is believed to be mediated, primarily by immune mechanisms, such as increasing cytokine production, improving dendritic cell maturation and function [15], promoting cytokine-induced killer cell (CIK) function [14], augmenting CTLs function [16] and enhancing the function of immunological effector cells in immunosuppressed mice [17]. Gl-PS can also inhibit the growth of vascular endothelial cells and the induction of vascular endothelial growth factor (VEGF) in human lung cancer cells [18]. Multidrug resistance (MDR) can also be reversed by Gl-PS through down-regulation of the expression of MDR-1 and MDR-associated protein 1 (MRP1) in an adriamycin (ADM)-resistant leukemic cell line (K562/ADM) [19]. Recently, it was demonstrated that the Gl-PS showed the effects on IEC-6 cell proliferation, migration and morphology of differentiation benefiting intestinal epithelium healing [20]. Unlike many other natural products, as our previous study has shown, Gl-PS has the promoting effects on B16F10 cells to activate lymphocytes [21]. Furthermore, the promoting effects of Gl-PS on B16F10 cells to activate lymphocytes may have the effect on cancer
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cells to induce efficient CTLs and sufficient granzyme B and porforin among CTLs, which remains unclear. Thus, the current study was designed to test this hypothesis.
and cultured for 5 days. Non-adherent cells were harvested as CTLs for assay. 2.5. Cell-mediated cytotoxicity assay
2. Materials and methods 2.1. Animals Male or female BALB/c (H-2d ) mice (grade II, certificate number SCXK2007-0001) and C57BL/6 (H-2b ) mice (grade II, certificate number SCXK2006-0008) were obtained from the Department of experimental animals of the health science centre of Peking University (Beijing, China). They were bred in the animal breeding facilities at Peking university health science centre (Beijing, China) under grade II conditions. All of the mice were used for experiments at 8 to 10 weeks of age. The use of mice was approved by the Ethics Committee. 2.2. Preparation of Ganoderma lucidum polysaccharides Gl-PS was isolated from a boiling water extract of Gl, and processed by ethanol precipitation, dialysis and protein depletion using the Sevag method, as described previously [4,15,16]. Gl-PS is a polysaccharide peptide with a molecular weight of 584,900 and the ratio of polysaccharides-to-peptides is 93.51:6.49. The polysaccharides consist of d-rhamnose, d-xylose, d-fructose, d-galactose, d-mannose, and d-glucose, with a molar ratio of 0.793:0.964:2.944:0.167:0.389:7.94, and are linked together by glycosidic linkages. The peptides contain 16 kinds of amino acids. Gl-PS is a hazel powder and was dissolved in serum-free RPMI1640 medium (Gibco BRL, Gaithersburg, MD, USA), then filtered through a 0.22 m filter and stored at 4 ◦ C. Gl-PS was further diluted to the indicated final concentration (200, 400, or 600 g/ml) and fetal bovine serum (FBS) was supplemented to 10% of the final concentration prior to each assay. 2.3. Preparation and culture of B16F10 cells Mouse B16F10 melanoma cells (H-2b ), a tumor cell line deficient in antigen presentation, were grown at 37 ◦ C in a humidified atmosphere containing 5% CO2 in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 g/ml). Unless otherwise specified, B16F10 cells were seeded at a density of 2 × 103 /well in 96-well culture plates, 2 × 104 /well in 24-well culture plates or 2 × 105 /well in 6-well culture plates, and after an overnight incubation for adherence, were treated with Gl-PS by replacement of medium with medium containing Gl-PS in the indicated concentration for a further 48 hours incubation. Cells were harvested for assay or continued for further experiments. RPMI-1640 medium instead of Gl-PS was used as a control. 2.4. Cytotoxic T lymphocytes induction by B16F10 cells treated with Ganoderma lucidum polysaccharides Splenic lymphocytes were stimulated by B16F10 melanoma cells treated with Gl-PS (B16F10-Gl-PS). B16F10 melanoma cells were cultured and treated with Gl-PS as described above. These B16F10-Gl-PS cells were pre-treated with 100 g/ml of mitomycin and washed thrice with RPMI-1640 medium before stimulation to remove residual mitomycin or Gl-PS. Mononuclear lymphocytes were isolated from splenocytes of BALB/c mice in a Ficoll-Urografin density gradient and counted by light microscopy. The mononuclear lymphocytes were seeded into the wells (1 × 107 cells/well in 24-well culture plates or 2 × 106 cells/well in 96-well culture plates) with B16F10-Gl-PS in the wells
Cell-mediated cytotoxicity was performed using CytoTox 96® non-radioactive cytotoxicity assay (Promega, San Luis Obispo, CA, USA) by measurement of lactate dehydrogenase (LDH) release according to the manufacturer’s protocol. CTLs served as effector cells (2 × 105 /well) and were incubated with B16F10 cells (5 × 103 /well) in 100 l of RPMI-1640 medium for 6 hours (effector cells: target cells = 40:1). The activities of LDH released into culture supernatants were measured. The cytotoxicity was determined by the following formula: cytotoxicity = (experimental – effector spontaneous – target spontaneous) × 100 / (target maximum – target spontaneous). 2.6. Immunocytochemistry CTLs smeared on slides were fixed with acetone for 5 minutes at room temperature. The endogenous peroxidase activity was quenched with 3% hydrogen peroxide. After blocking with 10% normal serum, goat polyclonal primary antibody against granzyme B or porforin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used at a 1:50 dilution and incubated overnight at 4 ◦ C. The next day, the HRP-labeled secondary antibody was applied for 1 hour and staining was finalized with a diaminobenzidine (DAB) solution to detect the target antigen. Slides were extensively washed with phosphate-buffered saline (PBS) between the different stages and counterstained with hematoxylin before mounting. Slides were examined under a light microscope. Replacement of primary antibody with PBS was used as a negative control. 2.7. SDS-polyacrylamide electrophoresis and Western blot The protein levels of granzyme B and porforin expressed in CTLs were determined by Western blot. The levels of total protein extracted from CTLs were determined with the Bradford assay. Equal amounts of protein (50 g) were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were subsequently preblocked in TBS containing 5% non-fat milk powder and then incubated with goat polyclonal anti-granzyme B or antiporforin antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:100 followed by peroxidase-conjugated rabbit anti-goat IgG antibody. The antigen-antibody complex was visualized with Western blotting luminol reagent (Santa Cruz Biotechnology, Inc.). The bands were quantified with a Gel Doc 2000 system and Quantity One software (BIO-RAD). 2.8. Tumor suppression assay in vivo B16F10-Gl-PS cells were washed three times with PBS and subcutaneously inoculated into syngenic C57BL/6 mice (2.5 × 105 cells/mouse). The visible or palpable tumors growing in the mice were monitored and recorded daily for 3 weeks. The latencies of the visible or palpable tumors in the mice were calculated 21 days after inoculation and the incidences of tumors 15 days after inoculation was also calculated. Tumor volumes were measured as well. 2.9. Statistical analysis The results are expressed as the mean (± SD) of quadruplicate experiments, except immunocytochemistry and statistical comparison between the experimental groups versus the control
L.-X. Sun et al. / Biomedicine & Preventive Nutrition 2 (2012) 113–118
was performed using one-way ANOVA, followed by the Dunnett’s t-test, except the incidence of tumors in vivo, for which the chi-square analysis was used. P values < 0.05 were considered significant.
3. Results 3.1. Cytotoxicity of cytotoxic T lymphocytes induced by B16F10-Ganoderma lucidum polysaccharides against B16F10 cells CTLs induced by B16F10-Gl-PS as effector cells exert their effects through cell-mediated cytotoxicity against B16F10 cells. It was shown by the LDH release assay that the cytotoxicity of these CTLs against B16F10 cells was significantly increased in those CTLs induced by B16F10 cells pre-treated with 200, 400, or 600 g/ml of Gl-PS (all P < 0.01, Fig. 1A) compared with the control.
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3.2. Production of granzyme B and porforin in cytotoxic T lymphocytes induced by B16F10-Ganoderma lucidum polysaccharides At the molecular level, with the exception of the death receptor pathway, the granule exocytosis pathway accounts for virtually all of the measurable contact-mediated cytotoxicity delivered by natural killer cells and CTLs [22,23]. Granzyme B and porforin are important components of the granule exocytosis pathway. Using immunocytochemistry techniques, it was shown that the expression of granzyme B (Fig. 2A) and porforin (Fig. 2B) induced in CTLs by B16F10 cells which had been pre-treated with various concentrations of Gl-PS was increased. The Western blot assay also showed that both the granzyme B and porforin in the CTLs, induced by B16F10 cells pre-treated with 400 g/ml of Gl-PS, were increased significantly compared with controls (both P < 0.01, Figs. 1B and C, B and D).
Fig. 1. Improved cytotoxicity in cytotoxic T lymphocytes with granzyme B and porforin and suppressed tumorigenesis of B16F10 melanoma. After B16F10 cells were treated with Ganoderma lucidum polysaccharides (Gl-PS) for 48 hours, followed by 100 g/ml of mitomycin pre-treatment and thrice washing with RPMI-1640, splenic mononuclear lymphocytes of BALB/c mice were seeded into the wells, and non-adherent lymphocytes as cytotoxic T lymphocytes were harvested after 5 days of incubation for assay of cytotoxicity by the lactose dehydrogenase release assay, as well as granzyme B and porforin by Western blot. The tumorigenic incidence in 15 days and latency in 21 days of B16F10 melanoma in vivo after inoculation of these B16F10 cells pre-incubated with Gl-PS in vitro for 48 hours were monitored. All experiments were performed in quadruplicate, except the tumorigenesis of B16F10 melanoma in vivo performed with ten mice per group. Error bars indicate standard deviation of the means. Asterisks indicate Dunnett t-test P-values < 0.05, compared with the control after one-way ANOVA. A. Mean percentages of cytotoxicities of cytotoxic T lymphocytes against B16F10 cells. B. Granzyme B and porforin in cytotoxic T lymphocytes detected by Western blot. C. Mean quantification in arbitrary units of granzyme B and (D) porforin in cytotoxic T lymphocytes relative to the -actin control evaluated by Western blot. E. Tumorigenic incidence of B16F10 melanoma in vivo in 15 days and (F) tumorigenic latency of B16F10 melanoma in vivo in 21 days after inoculation of these B16F10 cells pre-incubated with Gl-PS in vitro for 48 hours.
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Fig. 2. Improved granzyme B and porforin expression in cytotoxic T lymphocytes. After B16F10 cells were treated with Ganoderma lucidum polysaccharides (Gl-PS) for 48 hours, followed by 100 g/ml of mitomycin pre-treatment and thrice washing with RPMI-1640, splenic mononuclear lymphocytes of BALB/c mice were seeded into the wells, and non-adherent lymphocytes as cytotoxic T lymphocytes were harvested after 5 days of incubation for assay of granzyme B and porforin by immunocytochemistry. A. Granzyme B. B. Porforin.
L.-X. Sun et al. / Biomedicine & Preventive Nutrition 2 (2012) 113–118
3.3. Suppression against B16F10-Ganoderma lucidum polysaccharides cells in vivo in syngeneic C57BL/6 mice The ultimate purpose in boosting cancer cells to induce efficient CTLs and sufficient granzyme B and porforin is suppression or rejection of cancer cells in vivo. To distinguish the direct effects of Gl-PS on B16F10 cells from the effects of boosting host immune function, we pre-treated B16F10 cells with Gl-PS in vitro, then inoculated the cells into syngeneic C57BL/6 mice. It was shown that after treatment with 200 or 400 g/ml of Gl-PS, the incidence of tumorigenesis 15 days after inoculation was significantly decreased (both P < 0.05, Fig. 1E) and the latency of tumorigenesis 21 days after inoculation was significantly prolonged (both P < 0.05, Fig. 1F). Although the mean volumes of the tumors were less in the group treated with 200 or 400 g/ml of Gl-PS compared with the control, no statistical differences were demonstrated, likely reflecting the striking deviations (data not shown).
4. Discussion Based on the theory that the host immune system has the potential to control malignant cells, tumor immunotherapy has undergone significant development, but only limited clinical success has been achieved. One main reason for the gap, which exists between the theoretical and actual clinical efficacy of immunotherapy, is that tumor cells do not induce sufficient immune responses, such as efficient CTLs and sufficient granzyme B and porforin because of the weak immunogenicity of tumor cells. It is therefore important to devise ways to boost malignant cells to induce efficient CTLs and subsequently, sufficient granzyme B and porforin. Our previous studies [13–19,24] and studies by others [25,26] have demonstrated that Gl-PS possesses anti-tumor effects, chiefly by improving host immune function [5] It has been suggested that GlPS, with multiple known bioactivities, has the potential to boost tumor cells to induce efficient CTLs and sufficient granzyme B and porforin by which significant benefit on cancer immunotherapy can be achieved. Efficient induction of CTLs with sufficient granzyme B and porforin usually results in efficient cell-mediated cytotoxicity against target cells, which is the ultimate purpose of anti-tumor immunotherapy. As a result, the increased cytotoxicity mediated by the CTLs against B16F10 melanoma cells was achieved according to LDH release assay and the granzyme B and porforin were improved according to immunocytochemistry and Western blot assay. Between the two effector pathways (granule-mediated and death receptor-mediated pathways) used by CTLs, the granule exocytosis mechanism is the major immune effector mechanism of cytotoxicity for CTLs against target cells has become well-accepted since it was proposed in the 1980s [23,27,28]. With this pathway, mediators are delivered to the target cells, which elicit caspasedependent and -independent death pathways in the target cells. The critical mediators in this pathway include porforin (PFN) and granzymes. The granzymes are a family of serine proteases, of which granzyme B is the most potent member. Granzymes (or other granule components) act in cooperation with PFN to transduce the apoptotic signal. PFN inserts itself into the plasma membrane of the target cell, facilitating endocytosis of PFN and granzyme proteases. The subsequent translocation of pro-apoptotic granzymes into the cytoplasm [29–32] provides these proteases access to numerous protein substrates that promote apoptosis after cleavage, [33–37] followed by apoptosis in the target cells. Immunocytochemistry and Western blot have shown that after incubation with Gl-PS in certain concentration, B16F10 cells induced more sufficient production of granzyme B and porforin in CTLs, which play a role in cytotoxicity against tumor cells, resulted
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in more efficient CTLs. It was also shown in vivo that the incidence of tumorigenesis of the B16F10 cells pre-treated in vitro with GlPS was significantly decreased at 15 days and the latency of the tumorigenesis of the B16F10 cells pre-treated in vitro with Gl-PS at 21 days was significantly prolonged, demonstrating that the effects of Gl-PS on B16F10 cells suppressed the tumorigenicity of these cells in vivo in syngenic mice, presumably by improving the nature of B16F10 cells, wholly or in part, to induce efficient CTLs. With the accumulation of the increased cytotoxicity mediated by the CTLs against B16F10 melanoma cells, the increased granzyme B and porforin, and the decreased incidence of tumorigenesis and prolonged latency of the tumorigenesis in the B16F10 cells pre-treated in vitro with Gl-PS at 15 and 21 days of inoculation, respectively, the upmost effects were found in the concentration of 400 g/ml Gl-PS although the effects in concentrations nearest it (200 g/ml or 600 g/ml) varied in some extent probably because many procedures required in the experiment with random influences involved, showing Gl-PS with most powerful effect in certain appropriate concentration other than higher concentration with higher effect. It can be concluded that certain concentration of GlPS boosts B16F10 melanoma cells to induce more efficient CTLs and subsequently the sufficient granzyme B and PFN exhibit more efficacious cytotoxicity against B16F10 melanoma cells. Because weak immunogenicity in tumor cells can show stronger results in allogenic lymphocytes, allo-stimulation and allo-killer assay in vitro was used to get more detectable results. Although the syngenic assay in vitro is more convincing, allogenic assay with suitable control can also provide valuable result in some extent. Compared to the control, which was not treated with Gl-PS, even if Gl-PS treated B16F10 cells as a stimulus induced stronger mixed lymphocyte reaction, the improved immunogenicity and enhanced sensitivity to be recognized and rejected by the immune system were implied. To exclude the effect of residue Gl-PS on boosting lymphocytes directly, we performed preliminary experiments in which the B16F10 cells cultured with Gl-PS in concentrations indicated above for 30 minutes followed by mitomycin treatment and thrice washing with RPMI 1640 medium showed no obvious differences to the control. That the highest concentration of Gl-PS (600 g/ml) did not have the highest effect is another support. In consideration of the in vivo application, the concentrations of the Gl-PS used in this study may be a higher but there are no appreciable adverse effects. Gl-PS can also be used in topical application by interventional therapy. Otherwise, Gl-PS is still valuable for consideration of in vitro use, such as making a cancer vaccine for active immunotherapy or to treat tumor cells as an immunogen to stimulate adoptive effector cells for adoptive immunotherapy. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments The authors thank Professor Shuqian Lin of the Fuzhou Institute of Green Valley Bio-Pharm Technology for providing the Gl-PS. References [1] de Vries JF, von dem Borne PA, van Luxemburg-Heijs SA, Heemskerk MH, Willemze R, Falkenburg JH, et al. Differential activation of the death receptor pathway in human target cells induced by cytotoxic T lymphocytes showing different kinetics of killing. Haematologica 2007;92:1671–8. [2] Wowk ME, Trapani JA. Cytotoxic activity of the lymphocyte toxin granzyme B. Microbes Infect 2004;6:752–8. [3] Gross S, Walden P. Immunosuppressive mechanisms in human tumors: why we still cannot cure cancer? Immunol Lett 2008;116:7–14.
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Available online at
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Original article
Stronger cytotoxicity in CTLs with granzyme B and porforin was induced by Ganoderma lucidum polysaccharides acting on B16F10 cells Li-Xin Sun a,b , Zhi-Bin Lin a , Xin-Suo Duan b , Jie Lu b , Zhi-Hua Ge b,∗ , You-Xin Song b , Xue-Jun Li a , Min Li a , En-Hong Xing b , Ning Yang b , Wei-Dong Li a,∗ a b
Department of Pharmacology, Peking University Health Science Centre, School of Basic Medical Sciences, Beijing 100191, China Affiliated Hospital of Chengde Medical College, Chengde 067000, Hebei Province, China
a r t i c l e
i n f o
Article history: Received 24 December 2011 Accepted 25 January 2012 Keywords: Ganoderma lucidum polysaccharides Tumor CTL Granzyme B Porforin Cytotoxicity Tumorigenesis
a b s t r a c t Cytotoxic T lymphocytes (CTLs) exert cytotoxicity against tumor cells with granzyme B and porforin (two important components for cytotoxicity). One main reason for the limitation of clinical success in tumor immunotherapy is tumor cell inefficacy in inducing sufficient immune responses, such as efficient CTLs, and subsequently sufficient granzyme B and porforin activity because of weak immunogenicity of the tumor cells. It is therefore important to boost tumor cells to induce efficient CTLs and sufficient granzyme B and porforin. We suggest that Ganoderma lucidum polysaccharides (Gl-PS), with multiple bioactivities have this potential. We have shown that after incubation with Gl-PS, B16F10 melanoma cells, which are deficient in antigen presentation, promoted cytotoxicity of CTLs against B16F10 cells, induced more granzyme B and porforin in CTLs, decreased the in vivo incidence of tumorigenesis 15 days after inoculation and prolonged the latency of tumorigenesis 21 days after inoculation, demonstrating that the effects of Gl-PS on B16F10 cells induced stronger immune responses against tumor cells. © 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction The immune system has the potential to recognize and attack tumor cells by various means involving different effector cells. Lymphocytes play an important role in tumor control and cytotoxic T lymphocytes (CTLs) are most of the effector cells. There are two pathways (cell death receptor-mediated pathway and granule exocytosis pathway), by what CTLs effect cytotoxicity against the target cells [1]. Of the two pathways, the granule exocytosis pathway is the principal pathway and involves granzyme B and porforin, which are two of the most important components exerting cytotoxicity against target cells [2]. However, despite the potential to eliminate tumor cells, T cells fail to do so because cancer cells have various strategies to avoid rejection by the host immune system [3], thereby inefficient induction of CTLs and insufficient production of granzyme B and porforin in lymphocytes occurs in cancer patients. Indeed, cancer immunotherapy would benefit by boosting cancer cells to induce efficient CTLs and sufficient granzyme B and porforin. Ganoderma lucidum polysaccharides (Gl-PS) may be one means by which to boost cancer cells.
∗ Corresponding authors. Tel.: +86 10 8280 2798; fax: +86 10 8280 1686. E-mail addresses: [email protected] (Z.-H. Ge), [email protected] (W.-D. Li). 2210-5239/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.bionut.2012.01.001
Gl-PS is an extract from G. lucidum, which has been widely used in China for centuries to promote longevity and improve vigour, without appreciable adverse effects [4,5]. Like some other macromolecules [6–12], Gl-PS has anti-tumor and immunomodulatory effects. Over the past two decades, numerous studies have demonstrated the multiple biological activities of Gl-PS, including immunomodulatory and anti-tumor activities [5,13,14]. The anti-tumor effect of Gl-PS is believed to be mediated, primarily by immune mechanisms, such as increasing cytokine production, improving dendritic cell maturation and function [15], promoting cytokine-induced killer cell (CIK) function [14], augmenting CTLs function [16] and enhancing the function of immunological effector cells in immunosuppressed mice [17]. Gl-PS can also inhibit the growth of vascular endothelial cells and the induction of vascular endothelial growth factor (VEGF) in human lung cancer cells [18]. Multidrug resistance (MDR) can also be reversed by Gl-PS through down-regulation of the expression of MDR-1 and MDR-associated protein 1 (MRP1) in an adriamycin (ADM)-resistant leukemic cell line (K562/ADM) [19]. Recently, it was demonstrated that the Gl-PS showed the effects on IEC-6 cell proliferation, migration and morphology of differentiation benefiting intestinal epithelium healing [20]. Unlike many other natural products, as our previous study has shown, Gl-PS has the promoting effects on B16F10 cells to activate lymphocytes [21]. Furthermore, the promoting effects of Gl-PS on B16F10 cells to activate lymphocytes may have the effect on cancer
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cells to induce efficient CTLs and sufficient granzyme B and porforin among CTLs, which remains unclear. Thus, the current study was designed to test this hypothesis.
and cultured for 5 days. Non-adherent cells were harvested as CTLs for assay. 2.5. Cell-mediated cytotoxicity assay
2. Materials and methods 2.1. Animals Male or female BALB/c (H-2d ) mice (grade II, certificate number SCXK2007-0001) and C57BL/6 (H-2b ) mice (grade II, certificate number SCXK2006-0008) were obtained from the Department of experimental animals of the health science centre of Peking University (Beijing, China). They were bred in the animal breeding facilities at Peking university health science centre (Beijing, China) under grade II conditions. All of the mice were used for experiments at 8 to 10 weeks of age. The use of mice was approved by the Ethics Committee. 2.2. Preparation of Ganoderma lucidum polysaccharides Gl-PS was isolated from a boiling water extract of Gl, and processed by ethanol precipitation, dialysis and protein depletion using the Sevag method, as described previously [4,15,16]. Gl-PS is a polysaccharide peptide with a molecular weight of 584,900 and the ratio of polysaccharides-to-peptides is 93.51:6.49. The polysaccharides consist of d-rhamnose, d-xylose, d-fructose, d-galactose, d-mannose, and d-glucose, with a molar ratio of 0.793:0.964:2.944:0.167:0.389:7.94, and are linked together by glycosidic linkages. The peptides contain 16 kinds of amino acids. Gl-PS is a hazel powder and was dissolved in serum-free RPMI1640 medium (Gibco BRL, Gaithersburg, MD, USA), then filtered through a 0.22 m filter and stored at 4 ◦ C. Gl-PS was further diluted to the indicated final concentration (200, 400, or 600 g/ml) and fetal bovine serum (FBS) was supplemented to 10% of the final concentration prior to each assay. 2.3. Preparation and culture of B16F10 cells Mouse B16F10 melanoma cells (H-2b ), a tumor cell line deficient in antigen presentation, were grown at 37 ◦ C in a humidified atmosphere containing 5% CO2 in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 g/ml). Unless otherwise specified, B16F10 cells were seeded at a density of 2 × 103 /well in 96-well culture plates, 2 × 104 /well in 24-well culture plates or 2 × 105 /well in 6-well culture plates, and after an overnight incubation for adherence, were treated with Gl-PS by replacement of medium with medium containing Gl-PS in the indicated concentration for a further 48 hours incubation. Cells were harvested for assay or continued for further experiments. RPMI-1640 medium instead of Gl-PS was used as a control. 2.4. Cytotoxic T lymphocytes induction by B16F10 cells treated with Ganoderma lucidum polysaccharides Splenic lymphocytes were stimulated by B16F10 melanoma cells treated with Gl-PS (B16F10-Gl-PS). B16F10 melanoma cells were cultured and treated with Gl-PS as described above. These B16F10-Gl-PS cells were pre-treated with 100 g/ml of mitomycin and washed thrice with RPMI-1640 medium before stimulation to remove residual mitomycin or Gl-PS. Mononuclear lymphocytes were isolated from splenocytes of BALB/c mice in a Ficoll-Urografin density gradient and counted by light microscopy. The mononuclear lymphocytes were seeded into the wells (1 × 107 cells/well in 24-well culture plates or 2 × 106 cells/well in 96-well culture plates) with B16F10-Gl-PS in the wells
Cell-mediated cytotoxicity was performed using CytoTox 96® non-radioactive cytotoxicity assay (Promega, San Luis Obispo, CA, USA) by measurement of lactate dehydrogenase (LDH) release according to the manufacturer’s protocol. CTLs served as effector cells (2 × 105 /well) and were incubated with B16F10 cells (5 × 103 /well) in 100 l of RPMI-1640 medium for 6 hours (effector cells: target cells = 40:1). The activities of LDH released into culture supernatants were measured. The cytotoxicity was determined by the following formula: cytotoxicity = (experimental – effector spontaneous – target spontaneous) × 100 / (target maximum – target spontaneous). 2.6. Immunocytochemistry CTLs smeared on slides were fixed with acetone for 5 minutes at room temperature. The endogenous peroxidase activity was quenched with 3% hydrogen peroxide. After blocking with 10% normal serum, goat polyclonal primary antibody against granzyme B or porforin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used at a 1:50 dilution and incubated overnight at 4 ◦ C. The next day, the HRP-labeled secondary antibody was applied for 1 hour and staining was finalized with a diaminobenzidine (DAB) solution to detect the target antigen. Slides were extensively washed with phosphate-buffered saline (PBS) between the different stages and counterstained with hematoxylin before mounting. Slides were examined under a light microscope. Replacement of primary antibody with PBS was used as a negative control. 2.7. SDS-polyacrylamide electrophoresis and Western blot The protein levels of granzyme B and porforin expressed in CTLs were determined by Western blot. The levels of total protein extracted from CTLs were determined with the Bradford assay. Equal amounts of protein (50 g) were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were subsequently preblocked in TBS containing 5% non-fat milk powder and then incubated with goat polyclonal anti-granzyme B or antiporforin antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:100 followed by peroxidase-conjugated rabbit anti-goat IgG antibody. The antigen-antibody complex was visualized with Western blotting luminol reagent (Santa Cruz Biotechnology, Inc.). The bands were quantified with a Gel Doc 2000 system and Quantity One software (BIO-RAD). 2.8. Tumor suppression assay in vivo B16F10-Gl-PS cells were washed three times with PBS and subcutaneously inoculated into syngenic C57BL/6 mice (2.5 × 105 cells/mouse). The visible or palpable tumors growing in the mice were monitored and recorded daily for 3 weeks. The latencies of the visible or palpable tumors in the mice were calculated 21 days after inoculation and the incidences of tumors 15 days after inoculation was also calculated. Tumor volumes were measured as well. 2.9. Statistical analysis The results are expressed as the mean (± SD) of quadruplicate experiments, except immunocytochemistry and statistical comparison between the experimental groups versus the control
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was performed using one-way ANOVA, followed by the Dunnett’s t-test, except the incidence of tumors in vivo, for which the chi-square analysis was used. P values < 0.05 were considered significant.
3. Results 3.1. Cytotoxicity of cytotoxic T lymphocytes induced by B16F10-Ganoderma lucidum polysaccharides against B16F10 cells CTLs induced by B16F10-Gl-PS as effector cells exert their effects through cell-mediated cytotoxicity against B16F10 cells. It was shown by the LDH release assay that the cytotoxicity of these CTLs against B16F10 cells was significantly increased in those CTLs induced by B16F10 cells pre-treated with 200, 400, or 600 g/ml of Gl-PS (all P < 0.01, Fig. 1A) compared with the control.
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3.2. Production of granzyme B and porforin in cytotoxic T lymphocytes induced by B16F10-Ganoderma lucidum polysaccharides At the molecular level, with the exception of the death receptor pathway, the granule exocytosis pathway accounts for virtually all of the measurable contact-mediated cytotoxicity delivered by natural killer cells and CTLs [22,23]. Granzyme B and porforin are important components of the granule exocytosis pathway. Using immunocytochemistry techniques, it was shown that the expression of granzyme B (Fig. 2A) and porforin (Fig. 2B) induced in CTLs by B16F10 cells which had been pre-treated with various concentrations of Gl-PS was increased. The Western blot assay also showed that both the granzyme B and porforin in the CTLs, induced by B16F10 cells pre-treated with 400 g/ml of Gl-PS, were increased significantly compared with controls (both P < 0.01, Figs. 1B and C, B and D).
Fig. 1. Improved cytotoxicity in cytotoxic T lymphocytes with granzyme B and porforin and suppressed tumorigenesis of B16F10 melanoma. After B16F10 cells were treated with Ganoderma lucidum polysaccharides (Gl-PS) for 48 hours, followed by 100 g/ml of mitomycin pre-treatment and thrice washing with RPMI-1640, splenic mononuclear lymphocytes of BALB/c mice were seeded into the wells, and non-adherent lymphocytes as cytotoxic T lymphocytes were harvested after 5 days of incubation for assay of cytotoxicity by the lactose dehydrogenase release assay, as well as granzyme B and porforin by Western blot. The tumorigenic incidence in 15 days and latency in 21 days of B16F10 melanoma in vivo after inoculation of these B16F10 cells pre-incubated with Gl-PS in vitro for 48 hours were monitored. All experiments were performed in quadruplicate, except the tumorigenesis of B16F10 melanoma in vivo performed with ten mice per group. Error bars indicate standard deviation of the means. Asterisks indicate Dunnett t-test P-values < 0.05, compared with the control after one-way ANOVA. A. Mean percentages of cytotoxicities of cytotoxic T lymphocytes against B16F10 cells. B. Granzyme B and porforin in cytotoxic T lymphocytes detected by Western blot. C. Mean quantification in arbitrary units of granzyme B and (D) porforin in cytotoxic T lymphocytes relative to the -actin control evaluated by Western blot. E. Tumorigenic incidence of B16F10 melanoma in vivo in 15 days and (F) tumorigenic latency of B16F10 melanoma in vivo in 21 days after inoculation of these B16F10 cells pre-incubated with Gl-PS in vitro for 48 hours.
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Fig. 2. Improved granzyme B and porforin expression in cytotoxic T lymphocytes. After B16F10 cells were treated with Ganoderma lucidum polysaccharides (Gl-PS) for 48 hours, followed by 100 g/ml of mitomycin pre-treatment and thrice washing with RPMI-1640, splenic mononuclear lymphocytes of BALB/c mice were seeded into the wells, and non-adherent lymphocytes as cytotoxic T lymphocytes were harvested after 5 days of incubation for assay of granzyme B and porforin by immunocytochemistry. A. Granzyme B. B. Porforin.
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3.3. Suppression against B16F10-Ganoderma lucidum polysaccharides cells in vivo in syngeneic C57BL/6 mice The ultimate purpose in boosting cancer cells to induce efficient CTLs and sufficient granzyme B and porforin is suppression or rejection of cancer cells in vivo. To distinguish the direct effects of Gl-PS on B16F10 cells from the effects of boosting host immune function, we pre-treated B16F10 cells with Gl-PS in vitro, then inoculated the cells into syngeneic C57BL/6 mice. It was shown that after treatment with 200 or 400 g/ml of Gl-PS, the incidence of tumorigenesis 15 days after inoculation was significantly decreased (both P < 0.05, Fig. 1E) and the latency of tumorigenesis 21 days after inoculation was significantly prolonged (both P < 0.05, Fig. 1F). Although the mean volumes of the tumors were less in the group treated with 200 or 400 g/ml of Gl-PS compared with the control, no statistical differences were demonstrated, likely reflecting the striking deviations (data not shown).
4. Discussion Based on the theory that the host immune system has the potential to control malignant cells, tumor immunotherapy has undergone significant development, but only limited clinical success has been achieved. One main reason for the gap, which exists between the theoretical and actual clinical efficacy of immunotherapy, is that tumor cells do not induce sufficient immune responses, such as efficient CTLs and sufficient granzyme B and porforin because of the weak immunogenicity of tumor cells. It is therefore important to devise ways to boost malignant cells to induce efficient CTLs and subsequently, sufficient granzyme B and porforin. Our previous studies [13–19,24] and studies by others [25,26] have demonstrated that Gl-PS possesses anti-tumor effects, chiefly by improving host immune function [5] It has been suggested that GlPS, with multiple known bioactivities, has the potential to boost tumor cells to induce efficient CTLs and sufficient granzyme B and porforin by which significant benefit on cancer immunotherapy can be achieved. Efficient induction of CTLs with sufficient granzyme B and porforin usually results in efficient cell-mediated cytotoxicity against target cells, which is the ultimate purpose of anti-tumor immunotherapy. As a result, the increased cytotoxicity mediated by the CTLs against B16F10 melanoma cells was achieved according to LDH release assay and the granzyme B and porforin were improved according to immunocytochemistry and Western blot assay. Between the two effector pathways (granule-mediated and death receptor-mediated pathways) used by CTLs, the granule exocytosis mechanism is the major immune effector mechanism of cytotoxicity for CTLs against target cells has become well-accepted since it was proposed in the 1980s [23,27,28]. With this pathway, mediators are delivered to the target cells, which elicit caspasedependent and -independent death pathways in the target cells. The critical mediators in this pathway include porforin (PFN) and granzymes. The granzymes are a family of serine proteases, of which granzyme B is the most potent member. Granzymes (or other granule components) act in cooperation with PFN to transduce the apoptotic signal. PFN inserts itself into the plasma membrane of the target cell, facilitating endocytosis of PFN and granzyme proteases. The subsequent translocation of pro-apoptotic granzymes into the cytoplasm [29–32] provides these proteases access to numerous protein substrates that promote apoptosis after cleavage, [33–37] followed by apoptosis in the target cells. Immunocytochemistry and Western blot have shown that after incubation with Gl-PS in certain concentration, B16F10 cells induced more sufficient production of granzyme B and porforin in CTLs, which play a role in cytotoxicity against tumor cells, resulted
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in more efficient CTLs. It was also shown in vivo that the incidence of tumorigenesis of the B16F10 cells pre-treated in vitro with GlPS was significantly decreased at 15 days and the latency of the tumorigenesis of the B16F10 cells pre-treated in vitro with Gl-PS at 21 days was significantly prolonged, demonstrating that the effects of Gl-PS on B16F10 cells suppressed the tumorigenicity of these cells in vivo in syngenic mice, presumably by improving the nature of B16F10 cells, wholly or in part, to induce efficient CTLs. With the accumulation of the increased cytotoxicity mediated by the CTLs against B16F10 melanoma cells, the increased granzyme B and porforin, and the decreased incidence of tumorigenesis and prolonged latency of the tumorigenesis in the B16F10 cells pre-treated in vitro with Gl-PS at 15 and 21 days of inoculation, respectively, the upmost effects were found in the concentration of 400 g/ml Gl-PS although the effects in concentrations nearest it (200 g/ml or 600 g/ml) varied in some extent probably because many procedures required in the experiment with random influences involved, showing Gl-PS with most powerful effect in certain appropriate concentration other than higher concentration with higher effect. It can be concluded that certain concentration of GlPS boosts B16F10 melanoma cells to induce more efficient CTLs and subsequently the sufficient granzyme B and PFN exhibit more efficacious cytotoxicity against B16F10 melanoma cells. Because weak immunogenicity in tumor cells can show stronger results in allogenic lymphocytes, allo-stimulation and allo-killer assay in vitro was used to get more detectable results. Although the syngenic assay in vitro is more convincing, allogenic assay with suitable control can also provide valuable result in some extent. Compared to the control, which was not treated with Gl-PS, even if Gl-PS treated B16F10 cells as a stimulus induced stronger mixed lymphocyte reaction, the improved immunogenicity and enhanced sensitivity to be recognized and rejected by the immune system were implied. To exclude the effect of residue Gl-PS on boosting lymphocytes directly, we performed preliminary experiments in which the B16F10 cells cultured with Gl-PS in concentrations indicated above for 30 minutes followed by mitomycin treatment and thrice washing with RPMI 1640 medium showed no obvious differences to the control. That the highest concentration of Gl-PS (600 g/ml) did not have the highest effect is another support. In consideration of the in vivo application, the concentrations of the Gl-PS used in this study may be a higher but there are no appreciable adverse effects. Gl-PS can also be used in topical application by interventional therapy. Otherwise, Gl-PS is still valuable for consideration of in vitro use, such as making a cancer vaccine for active immunotherapy or to treat tumor cells as an immunogen to stimulate adoptive effector cells for adoptive immunotherapy. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments The authors thank Professor Shuqian Lin of the Fuzhou Institute of Green Valley Bio-Pharm Technology for providing the Gl-PS. References [1] de Vries JF, von dem Borne PA, van Luxemburg-Heijs SA, Heemskerk MH, Willemze R, Falkenburg JH, et al. Differential activation of the death receptor pathway in human target cells induced by cytotoxic T lymphocytes showing different kinetics of killing. Haematologica 2007;92:1671–8. [2] Wowk ME, Trapani JA. Cytotoxic activity of the lymphocyte toxin granzyme B. Microbes Infect 2004;6:752–8. [3] Gross S, Walden P. Immunosuppressive mechanisms in human tumors: why we still cannot cure cancer? Immunol Lett 2008;116:7–14.
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